Meter for use in an improved method of reducing interferences in an electrochemical sensor using two different applied potentials

ABSTRACT

The present invention is directed to an improved meter that utilizes a method of reducing the effects of interfering compounds in the measurement of analytes and more particularly to a method of reducing the effects of interfering compounds in a system wherein the test strip utilizes two or more working electrodes. In one embodiment of the present invention, a meter is described which applies a first potential to a first working electrode and a second potential, having the same polarity but a greater magnitude than the first potential, is applied to a second working electrode. The meter then measures the generated current and utilizes a predetermined algorithm to correct the measured current to compensate for the presence of interfering compounds in the sample.

PRIORITY

This application claims priority from Provisional Application No.60/516,252 filed Oct. 31, 2003, Provisional Application No. 60/558,728filed Mar. 31, 2004, and Provisional Application No. 60/558,424 filedMar. 31, 2004, which are incorporated herein by reference and to whichwe claim priority.

RELATED APPLICATIONS

The present invention is related to the following co-pending U.S.applications: U.S. patent application Ser. No. 10/977,154, filed on Oct.29, 2004; U.S. patent application Ser. No. 10/976,489, filed on Oct. 29,2004; U.S. patent application Ser. No. 10/977,292, filed on Oct. 29,2004; U.S. patent application Ser. No. 10/977,316, filed on Oct. 29,2004; and U.S. patent application Ser. No. 10/977,086, filed on Oct. 29,2004.

BACKGROUND OF INVENTION

Electrochemical glucose test strips, such as those used in the OneTouch®Ultra® whole blood testing kit, which is available from LifeScan, Inc.,are designed to measure the concentration of glucose in a blood samplefrom patients with diabetes. The measurement of glucose is based uponthe specific oxidation of glucose by the flavo-enzyme glucose oxidase.During this reaction, the enzyme becomes reduced. The enzyme isre-oxidized by reaction with the mediator ferricyanide, which is itselfreduced during the course or the reaction. These reactions aresummarized below.D-Glucose+GOx_((OX))→Gluconic acid+GOx_((RED))GOx_((RED))+2 Fe(CN)₆ ³⁻→GOx_((OX))+2 Fe(CN)₆ ⁴⁻

When the reaction set forth above is conducted with an applied potentialbetween two electrodes, an electrical current may be created by theelectrochemical re-oxidation of the reduced mediator ion (ferrocyanide)at the electrode surface. Thus, since, in an ideal environment, theamount of ferrocyanide created during the chemical reaction describedabove is directly proportional to the amount of glucose in the samplepositioned between the electrodes, the current generated would beproportional to the glucose content of the sample. A redox mediator,such as ferricyanide is a compound that exchanges electrons between aredox enzyme such as glucose oxidase and an electrode. As theconcentration of glucose in the sample increases, the amount of reducedmediator formed also increases, hence, there is a direct relationshipbetween current resulting from the re-oxidation of reduced mediator andglucose concentration. In particular, the transfer of electrons acrossthe electrical interface results in a flow of current (2 moles ofelectrons for every mole of glucose that is oxidized). The currentresulting from the introduction of glucose may, therefore, be referredto as the glucose current.

Because it can be very important to know the concentration of glucose inblood, particularly in people with Diabetes, meters have been developedusing the principals set forth above to enable the average person tosample and test their blood to determine the glucose concentration atany given time. The Glucose Current generated is monitored by the meterand converted into a reading of glucose concentration using a presetalgorithm that relates current to glucose concentration via a simplemathematical formula. In general, the meters work in conjunction with adisposable strip that includes a sample chamber and at least twoelectrodes disposed within the sample chamber in addition to the enzyme(e.g. glucose oxidase) and mediator (e.g. ferricyanide). In use, theuser pricks their finger or other convenient site to induce bleeding andintroduces a blood sample to the sample chamber, thus starting thechemical reaction set forth above.

In electrochemical terms, the function of the meter is two fold.Firstly, it provides a polarizing voltage (approximately 0.4 V in thecase of OneTouch® Ultra®) that polarizes the electrical interface andallows current flow at the carbon working electrode surface. Secondly,it measures the current that flows in the external circuit between theanode (working electrode) and the cathode (reference electrode). Themeter may, therefore be considered to be a simple electrochemical systemthat operates in two-electrode mode although, in practice, third and,even fourth electrodes may be used to facilitate the measurement ofglucose and/or perform other functions in the meter.

In most situations, the equation set forth above is considered to be asufficient approximation of the chemical reaction taking place on thetest strip and the meter reading a sufficiently accurate representationof the glucose content of the blood sample. However, under certaincircumstances and for certain purposes, it may be advantageous toimprove the accuracy of the measurement. For example, where a portion ofthe current measured at the electrode results from the presence of otherchemicals or compounds in the sample. Where such additional chemicals orcompounds are present, they may be referred to as interfering compoundsand the resulting additional current may be referred to as InterferingCurrents

Examples of potentially interfering chemicals (i.e. compounds found inphysiological fluids such as blood that may generate InterferingCurrents in the presence of an electrical field) include ascorbate,urate and acetaminophen (Tylenol™ or Paracetamol). One mechanism forgenerating Interfering Currents in an electrochemical meter formeasuring the concentration of an analyte in a physiological fluid (e.g.a glucose meter) involves the oxidation of one or more interferingcompounds by reduction of the enzyme (e.g. glucose oxidase). A furthermechanism for generating Interfering Currents in such a meter involvesthe oxidation of one or more interfering compounds by reduction of themediator (e.g. ferricyanide). A further mechanism for generatingInterfering Currents in such a meter involves the oxidation of one ormore interfering compounds at the working electrode. Thus, the totalcurrent measured at the working electrode is the superposition of thecurrent generated by oxidation of the analyte and the current generatedby oxidation of interfering compounds. Oxidation of interferingcompounds may be a result of interaction with the enzyme, the mediatoror may occur directly at the working electrode.

In general, potentially interfering compounds can be oxidized at theelectrode surface and/or by a redox mediator. This oxidation of theinterfering compound in a glucose measurement system causes the measuredoxidation current to be dependent on both the glucose and theinterfering compound. Therefore, if the concentration of interferingcompound oxidizes as efficiently as glucose and/or the interferingcompound concentration is significantly high relative to the glucoseconcentration, it may impact the measured glucose concentration.

The co-oxidization of analyte (e.g. glucose) with interfering compoundsis especially problematic when the standard potential (i.e. thepotential at which a compound is oxidized) of the interfering compoundis similar in magnitude to the standard potential of the redox mediator,resulting in a significant portion of the Interference Current beinggenerated by oxidation of the interfering compounds at the workingelectrode. Electrical current resulting from the oxidation ofinterfering compounds at the working electrode may be referred to asdirect interference current. It would, therefore, be advantageous toreduce or minimize the effect of the direct interference current on themeasurement of analyte concentration. Previous methods of reducing oreliminating direct interference current include designing test stripsthat prevent the interfering compounds from reaching the workingelectrode, thus reducing or eliminating the direct interference currentattributable to the excluded compounds.

One strategy for reducing the effects of interfering compounds thatgenerate Direct interference current is to place a negatively chargedmembrane on top of the working electrode. As one example, a sulfonatedfluoropolymer such as NAFION™ may be placed over the working electrodeto repel all negatively charged chemicals. In general, many interferingcompounds, including ascorbate and urate, have a negative charge, andthus, are excluded from being oxidized at the working electrode when thesurface of the working electrode is covered by a negatively chargedmembrane. However, because some interfering compounds, such asacetaminophen, are not negatively charged, and thus, can pass throughthe negatively charged membrane, the use of a negatively chargedmembrane will not eliminate the Direct interference current. Anotherdisadvantage of covering the working electrode with a negatively chargedmembrane is that commonly used redox mediators, such as ferricyanide,are negatively charged and cannot pass through the membrane to exchangeelectrons with the electrode. A further disadvantage of using anegatively charged membrane over the working electrode is the potentialto slow the diffusion of the reduced mediator to the working electrode,thus increasing the test time. A further disadvantage of using anegatively charged membrane over the working electrode is the increasedcomplexity and expense of manufacturing the test strips with anegatively charged membrane.

Another strategy that can be used to decrease the effects of DirectInterfering Currents is to position a size selective membrane on top ofthe working electrode. As one example, a 100 Dalton size exclusionmembrane such as cellulose acetate may be placed over the workingelectrode to exclude compounds having a molecular weight greater than100 Daltons. In this embodiment, the redox enzyme such as glucoseoxidase is positioned over the size exclusion membrane. Glucose oxidasegenerates hydrogen peroxide, in the presence of glucose and oxygen, inan amount proportional to the glucose concentration. It should be notedthat glucose and most redox mediators have a molecular weight greaterthan 100 Daltons, and thus, cannot pass through the size selectivemembrane. Hydrogen peroxide, however, has a molecular weight of 34Daltons, and thus, can pass through the size selective membrane. Ingeneral, most interfering compounds have a molecular weight greater than100 Daltons, and thus, are excluded from being oxidized at the electrodesurface. Since some interfering compounds have smaller molecularweights, and thus, can pass through the size selective membrane, the useof a size selective membrane will not eliminate the Direct interferencecurrent. A further disadvantage of using a size selective membrane overthe working electrode is the increased complexity and expense ofmanufacturing the test strips with a size selective membrane.

Another strategy that can be used to decrease the effects of Directinterference current is to use a redox mediator with a low redoxpotential, for example, a redox potential of between about −300 mV to+100 mV (vs a saturated calomel electrode). This allows the appliedpotential to the working electrode to be relatively low which, in turn,decreases the rate at which interfering compounds are oxidized by theworking electrode. Examples of redox mediators having a relatively lowredox potential include osmium bipyridyl complexes, ferrocenederivatives, and quinone derivatives. However, redox mediators having arelatively low potential are often difficult to synthesize, relativelyunstable and relatively insoluble.

Another strategy that can be used to decrease the effects of interferingcompounds is to use a dummy electrode in conjunction with the workingelectrode. The current measured at the dummy electrode may then besubtracted from the current measured at the working electrode in orderto compensate for the effect of the interfering compounds. If the dummyelectrode is bare (i.e. not covered by an enzyme or mediator), then thecurrent measured at the dummy electrode will be proportional to theDirect interference current and subtracting the current measured at thedummy electrode from the current measured at the working electrode willreduce or eliminate the effect of the direct oxidation of interferingcompounds at the working electrode. If the dummy electrode is coatedwith a redox mediator then the current measured at the dummy electrodewill be a combination of Direct interference current and interferencecurrent resulting from reduction of the redox mediator by an interferingcompound. Thus, subtracting the current measured at the dummy electrodecoated with a redox mediator from the current measured at the workingelectrode will reduce or eliminate the effect of the direct oxidation ofinterfering compounds and the effect of interference resulting fromreduction of the redox mediator by an interfering compound at theworking electrode. In some instances the dummy electrode may also becoated with an inert protein or deactivated redox enzyme in order tosimulate the effect of the redox mediator and enzyme on diffusion.Because it is preferable that test strips have a small sample chamber sothat people with diabetes do not have to express a large blood sample,it may not be advantageous to include an extra electrode whichincrementally increases the sample chamber volume where the extraelectrode is not used to measure the analyte (e.g. glucose). Further, itmay be difficult to directly correlate the current measured at the dummyelectrode to interference currents at the working electrode. Finally,since the dummy electrode may be coated with a material or materials(e.g. redox mediator) which differ from the materials used to cover theworking electrode (e.g. redox mediator and enzyme), test strips whichuse dummy electrodes as a method of reducing or eliminating the effectof interfering compounds in a multiple working electrode system mayincrease the cost and complexity of manufacturing the test strip.

Certain test strip designs which utilize multiple working electrodes tomeasure analyte, such as the system used in the OneTouch® Ultra® glucosemeasurement system are advantageous because the use of two workingelectrodes. In such systems, it would, therefore, be advantageous todevelop a meter for use with such test strips in the reducing oreliminating the effect of interfering compounds. More particularly, itwould be advantageous to develop a meter for use with such strips inreducing or eliminating the effect of interfering compounds withoututilizing a dummy electrode, an intermediate membrane or a redoxmediator with a low redox potential.

SUMMARY OF INVENTION

The present invention is directed to a meter for use in a method ofreducing the effects of interfering compounds in the measurement ofanalytes and more particularly to a method of reducing the effects ofinterfering compounds in a system wherein the test strip utilizes two ormore working electrodes. In one embodiment of the present invention, ameter is adapted to measure an analyte using a method where a firstpotential is applied to a first working electrode and a secondpotential, having the same polarity but a greater magnitude than thefirst potential, is applied to a second working electrode. The magnitudeof the second potential may also be less than the first potential for anembodiment where a reduction current is used to measure the analyteconcentration. In one embodiment, the first working electrode and secondworking electrode may be covered with an enzyme reagent and redoxmediator that are analyte specific. The first potential applied to thefirst working electrode is selected such that it is sufficient tooxidize reduced redox mediator in a diffusion limited manner while thesecond potential is selected to have a magnitude (i.e. absolute value)greater than the magnitude of the first potential, resulting in a moreefficient oxidation of at the second working electrode. In thisembodiment of the invention, the current measured by the meter at thefirst working electrode includes an analyte current and interferingcompound current while the current measured at the second workingelectrode includes an analyte overpotential current and an interferingcompound overpotential current. It should be noted that the analytecurrent and the analyte overpotential current both refer to a currentthat corresponds to the analyte concentration and that the current is aresult of a reduced mediator oxidation. In an embodiment of thisinvention, the relationship between the currents at the first workingelectrode and second working electrode may be defined by the followingequation,

$A_{1} = \frac{W_{2} - {YW}_{1}}{X - Y}$where A₁ is the analyte current at the first working electrode, W₁ isthe current measured at the first working electrode, W₂ is the currentmeasured at the second working electrode, X is an analyte dependentvoltage effect factor and Y is an interfering compound dependent voltageeffect factor. Using the equation set forth above, in a meter accordingto the present invention, it is possible to reduce the effect ofoxidation currents resulting from the presence of interfering compoundsand calculate a corrected current value that is more representative ofthe concentration of analyte in the sample being measured.

In one embodiment of the present invention, the concentration of glucosein a sample placed on a test strip can be calculated by placing thesample on a test strip that is inserted into a meter according to thepresent invention. In this embodiment, the test strip has a firstworking electrode and second working electrode and a referenceelectrode, at least the first working electrode and second workingelectrodes being coated with chemical compounds (e.g. an enzyme and aredox mediator) adapted to facilitate the oxidation of glucose and thetransfer of electrons from the oxidized glucose to the first workingelectrode and the second working electrode when a potential is appliedby a meter according to the present invention between the first workingelectrode and the reference electrode, and the second working electrodeand the reference electrode. In accordance with the present invention, afirst potential is applied by the meter between the first workingelectrode and the reference electrode, the first potential beingselected to have a magnitude sufficient to ensure that the magnitude ofthe current generated by oxidation of the glucose in the sample islimited only by factors other than applied voltage (e.g. diffusion). Inaccordance with the present invention, a second potential is applied bythe meter between the second working electrode and the referenceelectrode, the second potential being greater in magnitude than thefirst potential and, in one embodiment of the present invention, thesecond potential being selected to increase the oxidation of interferingcompounds at the second working electrode. In a further embodiment ofthe present invention, the meter may be programmed to use the followingequation to reduce the effect of oxidation current resulting from thepresence of interfering compounds on the current used to calculate theconcentration of glucose in the sample. In particular, the glucoseconcentration may be derived using a calculated current A_(1G) where:

$A_{1G} = {\frac{W_{2} - {YW}_{1}}{X_{G} - Y}.}$where A_(1G) is a glucose current, W₁ is the current measured at thefirst working electrode, W₂ is the current measured at the secondworking electrode, X_(G) is a glucose dependent voltage effect factorand Y is an interfering compound dependent voltage effect factor.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is an exploded perspective view of a test strip embodiment foruse in the present invention.

FIG. 2 is a schematic view of a meter and strip for use in the presentinvention.

FIG. 3 is a hydrodynamic voltammogram illustrating the dependence ofapplied voltage with measured current.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a meter for use with a test stripwhich utilize the method described herein to measure analyte and, moreparticularly, to a meter as illustrated in FIG. 2 which is programmed inaccordance with the method described herein.

While the present invention is particularly adapted to the measurementof glucose concentration in blood, it will be apparent to those of skillin the art that the method described herein may be adapted to improvethe selectivity of other systems used for the electrochemicalmeasurement of analytes in physiological fluids. Examples of systemsthat may be adapted to improve selectivity using the method according tothe present invention include electrochemical sensors used to measurethe concentration of lactate, lactate, alcohol, cholesterol, aminoacids, choline, and fructosamine in physiological fluids. Examples ofphysiological fluids that may contain such analytes include blood,plasma, serum, urine, and interstitial fluid. It will further beunderstood that, while the method of the present invention is describedin an electrochemical system where the measured current is produced byoxidation, the invention would be equally applicable to a system whereinthe measured current is produced by reduction.

The present invention is directed to a method for improving theselectivity of an electrochemical measuring system that is particularlyadapted for use in a blood glucose measurement system. Moreparticularly, the present invention is directed to a method forimproving the selectivity of a blood glucose measurement system bypartially or wholly correcting for the effect of the direct interferencecurrent. Selectivity in such systems being a measure of the ability ofthe system to accurately measure the glucose concentration in a sampleof physiological fluid which includes one or more compounds which createan interfering current. Improvement of selectivity thus reduces thecurrent generated at the working electrode by the presence ofinterfering compounds (i.e. compounds other than glucose which oxidizeto generate interfering current) and makes the measured current morerepresentative of the glucose concentration. In particular, the measuredcurrent may be a function of the oxidation of interfering compoundscommonly found in physiological fluids such as, for example,acetaminophen (Tylenol™ or Paracetamol), ascorbic acid, bilirubin,dopamine, gentisic acid, glutathione, levodopa, methyldopa, tolazimide,tolbutamide and uric acid. Such interfering compounds may be oxidizedby, for example, reacting chemically with the redox mediator or byoxidizing at the electrode surface.

In a perfectly selective system, there would be no oxidation currentgenerated by any interfering compound and the entire oxidation currentwould be generated by oxidation of glucose. However, if oxidation ofinterfering compounds and the resulting oxidation current cannot beavoided the present invention describes a method of removing some or allof the effect of interfering compounds by quantifying the proportion ofthe overall oxidation current generated by the interfering compounds andsubtracting that quantity from the overall oxidation current. Inparticular, in a method according to the present invention, using a teststrip that includes first working electrode and second workingelectrode, two different potentials are applied and the oxidationcurrent generated at each of the working electrodes is measure used toestimate the respective oxidation current proportions for both theglucose and interfering compounds.

In one embodiment of a method according to the present invention, a teststrip is used which includes a sample chamber containing a first workingelectrode, a second working electrode, and a reference electrode. Thefirst working electrode, the second working electrode and the referenceelectrodes are covered by glucose oxidase (the enzyme) and aFerricyanide (the redox mediator). When a sample of blood (thephysiological fluid) is placed in the sample chamber, the glucoseoxidase is reduced by glucose in the blood sample generating gluconicacid. The gluconic acid is then oxidized by reduction of theFerricyanide to Ferrocyanide, yielding a reduced redox mediator with aconcentration proportional to the glucose concentration. An example of atest strip that may be suitable for use in a method according to thepresent invention is the OneTouch® Ultra® test strip sold by LifeScan,Inc. of Milpitas, Calif. Other suitable strips are described ininternational publication WO 01/67099A1 and WO 1/73124A2.

In one embodiment of a method according to the present invention a firstpotential is applied to a first working electrode and a second potentialis applied to the second working electrode. In this embodiment, thefirst potential is selected to be in a range in which the glucosecurrent response is relatively insensitive to the applied potential andthus the magnitude of the glucose current at the first working electrodeis limited by the amount of reduced redox mediator diffusing to thefirst working electrode. It should be noted that glucose is not directlyoxidized at a working electrode, but instead is indirectly oxidizedthrough using a redox enzyme and a redox mediator. In the description ofthe present invention, the glucose current refers to an oxidation ofreduced redox mediator that correlates to the gluocose concentration. Inan embodiment of the present invention where ferri/ferrocyanide is theredox mediator and carbon is the working electrode, the first potentialmay range from about 0 millivolts to about 500 millivolts, and morepreferably from about 385 millivolts to about 415 millivolts, and yeteven more preferably may range from about 395 to 405 mV. A secondpotential is applied to a second working electrode such that the secondpotential is greater than the first potential. Where the appliedpotential is greater than the potential needed to oxidize the glucose.In an embodiment of the present invention where ferri/ferrocyanide isthe redox mediator and carbon is the working electrode, the secondpotential may range from about 50 millivolts to about 1000 millivolts,and more preferably from about 420 millivolts to about 1000 millivolts.

Because the glucose current does not increase or increases onlyminimally with increasing potential, the glucose current at the secondworking electrode should be substantially equal to the glucose currentat the first working electrode, even though the potential at the secondworking electrode is greater than the potential at the first electrode.Thus, any additional current measured at the second working electrodemay be attributed to the oxidation of interfering compounds. In otherwords, the higher potential at the second working electrode should causea glucose overpotenital current to be measured at the second workingelectrode which is equal or substantially equal in magnitude to theglucose current at the first working electrode because the firstpotential and second potential are in a limiting glucose current rangewhich is insensitive to changes in applied potential. However, inpractice, other parameters may have an impact on the measured current,for example, where a higher potential is applied to the second workingelectrode, there is often a slight increase in the overall current atthe second working electrode as a result of an IR drop or capacitiveeffects. When an IR drop (i.e. uncompensated resistance) is present inthe system, a higher applied potential causes an increase in themeasured current magnitude. Examples of IR drops may be the nominalresistance of the first working electrode, second working electrode, thereference electrode, the physiological fluid between the workingelectrode and the reference electrode. In addition, the application of ahigher potential results in the formation of a larger ionic double layerwhich forms at the electrode/liquid interface, increasing the ioniccapacitance and the resulting current at either the firstworkingelectrode or second working electrode.

In order to determine the actual relationship between the glucosecurrent measured at the first working electrode and the second workingelectrode, it is necessary to develop a suitable equation. It should benoted that the glucose current at the second working electrode may alsobe referred to as a glucose overpotential current. A directlyproportional relationship between the glucose current and the glucoseoverpotential current may be described by the following equation.X _(G) ×A _(1G) =A _(2G)  (eq 1)where X_(G) is a glucose dependent voltage effect factor, A_(1G) is theglucose current at the first working electrode and A_(2G) is the glucosecurrent at the second working electrode.

In an embodiment of the present invention, where ferri/ferrocyanide isthe redox mediator and carbon is the working electrode, the voltageeffect factor X_(G) for glucose may be expected to be between about 0.95any about 1.1. In this embodiment of the invention, higher potentials donot have a significant impact on the glucose oxidation current becausethe redox mediator (ferrocyanide) has fast electron transfer kineticsand reversible electron transfer characteristics with the workingelectrode. Because the glucose current does not increase with increasingpotential after a certain point, the glucose current may be said to besaturated or in a diffusion limited regime.

In the embodiment of the present invention described above, glucose isindirectly measured by oxidizing ferrocyanide at the working electrodeand where the ferrocyanide concentration is directly proportional to theglucose concentration. The standard potential (E^(o)) value for aparticular electrochemical compound is a measure of that compound'sability to exchange electrons with other chemical compounds. In themethod according to the present invention, the potential at the firstworking electrode is selected to be greater than the standard potential(E^(o)) of the redox mediator. Because the first potential is selectedsuch that it is sufficiently greater than the E^(o) value of the redoxcouple, the oxidation rate does not increase substantially as theapplied potential increases. Thus, applying a greater potential at thesecond working electrode will not increase the oxidation at the secondworking electrode and any increased current measured at the higherpotential electrode must be due to other factors, such as, for example,oxidation of interfering compounds.

FIG. 3 is a hydrodynamic voltammogram illustrating the dependence ofapplied voltage with measured current where ferri/ferrocyanide is theredox mediator and carbon is the working electrode. Each data point onthe graph represents at least one experiment where a current is measured5 seconds after applying a voltage across a working electrode and areference electrode. FIG. 3 shows that the current forms an onset of aplateau region at about 400 mV because the applied voltage issufficiently greater than of the E^(o) value of ferrocyanide. Thus, asillustrated in FIG. 3, as the potential reaches approximately 400 mV,the glucose current becomes saturated because the oxidation offerrocyanide is diffusion limited (i.e. the diffusion of ferrocyanide tothe working electrode limits the magnitude of the measured current andis not limited by the electron transfer rate between ferrocyanide andthe electrode).

In general, current generated by the oxidation of interfering compoundsis not saturated by increases in applied voltage and shows a muchstronger dependence on applied potential than current generated byoxidation of ferrocyanide (the ferrocyanide having been generated fromthe interaction of glucose with the enzyme and the enzyme withferrocyanide. Typically, interfering compounds have slower electrontransfer kinetics than redox mediators (i.e. ferrocyanide). Thisdifference is ascribed to the fact that most interfering compoundsundergo an inner sphere electron transfer pathway as opposed to thefaster outer sphere electron transfer pathway of ferrocyanide. A typicalinner sphere electron transfer requires a chemical reaction to occur,such as a hydride transfer, before transferring an electron. Incontrast, an outer sphere electron transfer does not require a chemicalreaction before transferring an electron. Therefore, inner sphereelectron transfer rates are typically slower than outer sphere electrontransfers because they require an additional chemical reaction step. Theoxidation of ascorbate to dehydroascorbate is an example of an innersphere oxidation that requires the liberation of two hydride moieties.The oxidation of ferrocyanide to ferricyanide is an example of an outersphere electron transfer. Therefore, the current generated byinterfering compounds generally increases when testing at a higherpotential.

A relationship between an interfering compound current at the firstworking electrode and an interfering compound overpotential current atthe second working electrode can be described by the following equation,Y×I ₁ =I ₂  (eq 2)where Y is an interfering compound dependent voltage effect factor, I₁is the interfering compound current, and I₁₂ is the interfering compoundoverpotential current. Because the interfering compound voltage effectfactor Y is dependent upon a number of factors, including, theparticular interfering compound or compounds of concern and the materialused for the working electrodes, calculation of a particular interferingcompound dependent voltage effect factor for a particular system, teststrip, analyte and interfering compound or compounds may requireexperimentation to optimize the voltage effect factor for thosecriteria. Alternatively, under certain circumstances, appropriatevoltage effect factors may be derived or described mathematically.

In an embodiment of the present invention where ferri/ferrocyanide isthe redox mediator and carbon is the working electrode, the interferingcompound dependent voltage effect factor Y could be mathematicallydescribed using the Tafel equation for I₁ and I₂,

$\begin{matrix}{I_{1} = {a^{\prime}{\exp\left( \frac{\eta_{1}}{b^{\prime}} \right)}}} & \left( {{eq}\mspace{20mu} 2a} \right) \\{I_{2} = {a^{\prime}{\exp\left( \frac{\eta_{2}}{b^{\prime}} \right)}}} & \left( {{eq}\mspace{20mu} 2b} \right)\end{matrix}$where η₁=E₁−E^(o), η₂=E₂−E^(o), b′ is a constant depending of thespecific electroactive interfering compound, E₁ is the first potential,and E₂ is the second potential. The value of E^(o) (the standardpotential of a specific interfering compound) is not important becauseit is canceled out in the calculation of Δη. Equations 2, 2a, 2b can becombined and rearranged to yield the following equation,

$\begin{matrix}{Y = {\exp\left( \frac{\Delta\eta}{b^{\prime}} \right)}} & \left( {{eq}\mspace{14mu} 2c} \right)\end{matrix}$where Δη=E₁−E₂. Equation 2c provides a mathematical relationshipdescribing the relationship between Δη (i.e. the difference between thefirst potential and the second potential) and the interfering compounddependent voltage effect factor Y. In an embodiment of the presentinvention, Y may range from about 1 to about 100, and more preferablybetween about 1 and 10. In an embodiment of this invention, theinterfering compound dependent voltage effect factor Y may be determinedexperimentally for a specific interfering compound or combination ofinterfering compounds. It should be noted that the interfering compounddependent voltage effect factor Y for interfering compounds is usuallygreater than voltage effect factor X_(G) for glucose. As the followingsections will describe, the mathematical relationship of a) theinterfering compound current I₁ and the interfering compoundoverpotential current I₂; and b) the glucose current A_(1G) and theglucose overpotential current A_(2G) will allow a glucose algorithm tobe proposed which will reduce the effects of interfering compounds formeasuring glucose.

In an embodiment of the present invention, an algorithm was developed tocalculate a corrected glucose current (i.e. A_(1G) and A_(2G)) which isindependent of interferences. After dosing a sample onto a test strip, afirst potential is applied to the first working electrode and a secondpotential is applied to the second working electrode. At the firstworking electrode, a first current is measured which can be described bythe following equation,W ₁ =A _(1G) +I ₁  (eq 3)where W₁ is the first current at the first working electrode. In otherwords, the first current includes a superposition of the glucose currentA_(1G) and the interfering compound current I₁. More specifically, theinterfering compound current may be a direct interfering current whichhas been described hereinabove. At the second working electrode, asecond current is measured at the second potential or overpotentialwhich can be described by the following equation,W ₂ =A _(2G) +I ₂  (eq 4)where W₂ is the second current at the second working electrode, A_(2G)is the glucose overpotential current measured at the second potential,and I₂ is the interfering compound overpotential current measured at thesecond potential. More specifically, the interfering compoundoverpotential current may be a Direct Interfering compound Current whichhas been described hereinabove. Using the previously described 4equations (eq's 1 to 4) which contain 4 unknowns (A_(1G), A_(2G), I₁,and I2), it is now possible to calculate a corrected glucose currentequation which is independent of interfering compounds.

As the first step in the derivation, A_(2G) from eq 1 and I₂ from eq 2can be substituted into eq 4 to give the following eq 5.W ₂ =X _(G) A _(1G) +YI ₁  (eq 5)Next, eq 3 is multiplied by interfering compound dependent voltageeffect factor Y for interfering compounds to give eq 6.YW ₁ =Y A _(1G) +YI ₁  (eq 6)Eq 5 can now be subtracted from eq 6 to give the following form shown ineq 7W ₂ −YW ₁ =X _(G) A _(1G) −Y A _(1G)  (eq 7)Eq 7 can now be rearranged to solve for the corrected glucose currentA_(1G) measured at the first potential as shown in eq 8.

$\begin{matrix}{A_{1G} = \frac{W_{2} - {YW}_{1}}{X_{G} - Y}} & \left( {{eq}\mspace{14mu} 8} \right)\end{matrix}$Eq 8 outputs a corrected glucose current A_(1G) which removes theeffects of interferences requiring only the current output of the firstworking electrode and second working electrode (eg W₁ and W₂), glucosedependent voltage effect factors X_(G,) and interfering compounddependent voltage effect factor Y for interfering compounds.

A glucose meter containing electronics is electrically interfaced with aglucose test strip to measure the current from W₁ and W₂. In oneembodiment of the present invention, X_(G) and Y may be programmed intothe glucose meter as read only memory. In another embodiment of thepresent invention, X_(G) and Y may be transferred to the meter via acalibration code chip. The calibration code chip would have in itsmemory a particular set of values for X_(G) and Y which would becalibrated for a particular lot of test strips. This would account fortest strip lot-to-lot variations that may occur in X_(G) and Y.

In another embodiment of the present invention, the corrected glucosecurrent in eq 8 may be used by the meter only when a certain thresholdis exceeded. For example, if W₂ is about 10% or greater than W₁, thenthe meter would use eq 8 to correct for the current output. However, ifW₂ is about 10% or less than W₁, the interfering compound concentrationis low and thus the meter can simply take an average current valuebetween W₁ and W₂ to improve the accuracy and precision of themeasurement. Instead of simply averaging the current of W₁ and W₂, amore accurate approach may be to average W₁ with

$\frac{W_{2}}{X_{G}}$where the glucose dependent voltage effect factor X_(G) is taken intoaccount (note

$\frac{W_{2}}{X_{G}}$approximately equals A_(1G) according to eq 1 and 4 when I₂ is low). Thestrategy of using eq 8 only under certain situations where it is likelythat a significant level of interferences are in the sample mitigatesthe risk of overcorrecting the measured glucose current. It should benoted that when W₂ is sufficiently greater than W₁ by a large amount(e.g. about 100% or more), this is an indicator of having an unusuallyhigh concentration of interferences. In such a case, it may be desirableto output an error message instead of a glucose value because a veryhigh level of interfering compounds may cause a breakdown in theaccuracy of eq 8.

The following sections will describe a possible test strip embodimentwhich may be used with the proposed algorithm of the present inventionas shown in eq 8. FIG. 1 is an exploded perspective view of a test stripwhich may be used in the present invention. Test strip 600 includes sixlayers disposed upon a base substrate 5. These six layers are aconductive layer 50, an insulation layer 16, a reagent layer 22, anadhesive layer 60, a hydrophilic layer 70, and a top layer 80. Teststrip 600 may be manufactured in a series of steps wherein theconductive layer 50, insulation layer 16, reagent layer 22, adhesivelayer 60 are deposited on base substrate 5 using, for example, a screenprinting process. Hydrophilic layer 70 and top layer 80 may be deposedfrom a roll stock and laminated onto base substrate 5. The fullyassembled test strip 600 forms a sample receiving chamber that canaccept a blood sample so that it can be analyzed.

Conductive layer 50 includes reference electrode 10, first workingelectrode 12, second working electrode 14, a first contact 13, a secondcontact 15, a reference contact 11, and a strip detection bar 17.Suitable materials which may be used for the conductive layer are Au,Pd, Ir, Pt, Rh, stainless steel, doped tin oxide, carbon, and the like.Preferably, the material for the conductive layer may be a carbon inksuch as those described in U.S. Pat. No. 5,653,918.

Insulation layer 16 includes cutout 18 which exposes a portion ofreference electrode 10, first working electrode 12, and second workingelectrode 14 which can be wetted by a liquid sample. As a non-limitingexample, insulation layer (16 or 160) may be Ercon E6110-116 Jet BlackInsulayer Ink which may be purchased from Ercon, Inc.

Reagent layer 22 may be disposed on a portion of conductive layer 50 andinsulation layer 16. In an embodiment of the present invention, reagentlayer 22 may include chemicals such as a redox enzyme and redox mediatorwhich selectivity react with glucose. During this reaction, aproportional amount of a reduced redox mediator can be generated thatthen can be measured electrochemically so that a glucose concentrationcan be calculated. Examples of reagent formulations or inks suitable foruse in the present invention can be found in U.S. Pat. Nos. 5,708,247and 6,046,051; published international applications WO01/67099 andWO01/73124, all of which are incorporated by reference herein.

Adhesive layer 60 includes first adhesive pad 24, second adhesive pad26, and third adhesive pad 28. The side edges of first adhesive pad 24and second adhesive pad 26 located adjacent to reagent layer 22 eachdefine a wall of a sample receiving chamber. In an embodiment of thepresent invention, the adhesive layer may comprise a water based acryliccopolymer pressure sensitive adhesive which is commercially availablefrom Tape Specialties LTD in Tring, Herts, United Kingdom (part#A6435).

Hydrophilic layer 70 includes a distal hydrophilic pad 32 and proximalhydrophilic pad 34. As a non-limiting example, hydrophilic layer 70 be apolyester having one hydrophilic surface such as an anti-fog coatingwhich is commercially available from 3M. It should be noted that bothdistal hydrophilic film 32 and proximal hydrophilic film 34 are visiblytransparent enabling a user to observe a liquid sample filling thesample receiving chamber.

Top layer 80 includes a clear portion 36 and opaque portion 38. Toplayer 80 is disposed on and adhered to hydrophilic layer 70. As anon-limiting example, top layer 40 may be a polyester. It should benoted that the clear portion 36 substantially overlaps proximalhydrophilic pad 32 that allows a user to visually confirm that thesample receiving chamber is sufficiently filled. Opaque portion 38 helpsthe user observe a high degree of contrast between a colored fluid suchas, for example, blood within the sample receiving chamber and theopaque section of the top film.

FIG. 2 is a simplified schematic showing a meter 500 interfacing with atest strip 600. Meter 500 has three electrical contacts that form anelectrical connection to first working electrode 12, second workingelectrode 14, and reference electrode 10. In particular connector 101connects voltage source 103 to first working electrode 12, connector 102connects voltage source 104 to second working electrode 14 and commonconnector 100 connects voltage source 103 and 104 to reference electrode10. When performing a test, voltage source 103 in meter 500 applies afirst potential E₁ between first working electrode 12 and referenceelectrode 10 and voltage source 104 applies a second potential E₂between second working electrode 14 and reference electrode 10. A sampleof blood is applied such that first working electrode 12, second workingelectrode 14, and reference electrode 10 are covered with blood. Thiscauses reagent layer 22 to become hydrated, which generates ferrocyanidein an amount proportional to the glucose and/or interfering compoundconcentration present in the sample. After about 5 seconds from thesample application, meter 500 measures an oxidation current for bothfirst working electrode 12 and second working electrode 14. In a meteraccording to the present invention, the values of E₁ and E₂ aredetermined in accordance with the previously described method and thealgorithms described herein may be used to calculate an analyte currentin accordance with a method according to the present invention.

In the previously described first and second test strip embodiments, thefirst working electrode 12 and second working electrode 14 had the samearea. It should be noted that the present invention is not limited totest strips having equal areas. For alternative embodiments to thepreviously described strips where the areas are different, the currentoutput for each working electrode must be normalized for area. Becausethe current output is directly proportional to area, the terms withinEquation 1 to Equation 8 may be in units of amperes (current) or inamperes per unit area (i.e. current density).

It will be recognized that equivalent structures may be substituted forthe structures illustrated and described herein and that the describedembodiment of the invention is not the only structure which may beemployed to implement the claimed invention. In addition, it should beunderstood that every structure described above has a function and suchstructure can be referred to as a means for performing that function.While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to hose skilled inthe art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A meter for use with a test strip in the detection of analytes, saidmeter comprising: a first connector for connecting said meter to a firstworking electrode on a test strip; a second connector for connectingsaid meter to a second working electrode on said test strip; a commonconnector for connecting said meter to a reference electrode on saidtest strip; a first voltage source connected between said firstconnector and said common connector; and a second voltage sourceconnected between said second connector and said common connector;wherein said first and second voltage sources generate the firstpotential at said first connector and the second potential at saidsecond connector when said test strip is inserted into said meter and asample is applied to said test strip, wherein said first and secondvoltages have the same polarity; wherein said meter measures a firstcurrent value at said first connector and a second current value at saidsecond connector at a predetermined time after said test strip isinserted and sample applied; wherein the meter is configured to generatea preselected first potential at the first connector using the firstvoltage source, the first potential sufficient to ensure a magnitude ofcurrent at the first working electrode that is diffusion limited andinsensitive to applied potential; wherein the meter is also configuredto generate a preselected second potential at the second connector usingthe second voltage source, the magnitude of the second potential beinggreater than the magnitude of the first potential; and wherein the meteris configured to calculate a corrected value of analyte current usingthe following formula: $A_{1} = \frac{W_{2} - {YW}_{1}}{X - Y}$ where:A₁ is said corrected analyte current; W₁ is the current at said firstconnector measured at said predetermined time; W₂ is the currentmeasured at said second connector at said predetermined time; X is ananalyte dependent voltage effect factor; and Y is an interferingcompound dependent voltage effect factor.